Site-specific incorporation of 19F-nuclei at protein C-terminus to probe allosteric conformational transitions of metalloproteins

Allosteric conformational change is an important paradigm in the regulation of protein function, which is typically triggered by the binding of small cofactors, metal ions or protein partners. Here, we found those conformational transitions can be effectively monitored by ¹⁹F NMR, facilitated by a site-specific ¹⁹F incorporation strategy at the protein C-terminus using asparaginyl endopeptidase (AEP). Three case studies show that C-terminal ¹⁹F-nuclei can reveal protein dynamics not only adjacent but also distal to C-terminus, including those occurring in a hemoprotein neuroglobin (Ngb), calmodulin (CaM), and a cobalt metalloregulator (CoaR) responding to both cobalt and tetrapyrrole. In Ngb, the heme orientation disorder is affected by missense mutations that perturb backbone rigidity or surface charges close to the heme axial ligands. In CaM, the C-terminal ¹⁹F-nuclei is an ideal probe for detecting the binding states of Ca²⁺, peptides and inhibitors. Furthermore, multiple ¹⁹F-moieties were incorporated into the two domains of CoaR, revealing the intrinsically disordered C-terminal metal binding tail might be an allosteric conformational switch to maintain cobalt homeostasis and balance corrinoid biosynthesis. This study demonstrates that the AEP-based ¹⁹F-modification strategy can be applied to various targets to study allosteric regulation, especially for those biological processes modulated by the protein C-terminus.